The present invention relates to a method for producing heterologous glycosylated proteins in non-animal eukaryotic cells such as in transformed bryophyte, yeast, ciliate or algae cells. In particular, the method relates to a method for producing glycosylated proteins comprising animal glycosylation patterns—comprising sialic acid residues—, such as pharmaceutical proteins for use in mammals, e.g. humans, in bryophyte cells such as those of Physcomitrella patens, the genetic material required therefore, such as DNA and RNA, vectors, host cells, methods of introducing genetic material there into, and uses thereof. Furthermore, the present invention relates to novel polypeptides and proteins obtained by the method according to the invention. Moreover, the present invention provides a method of producing sialic acid or CMP-sialic acid in a transformed non-mammalian eukaryotic cell, tissue or organism.
Plants are appropriate organisms for the production of a wide range of recombinant proteins (Ma et al. (2003) Nat Gen 4, 794-805). In terms of pharmaceutical proteins for use in mammals, including humans, post-translational modifications, such as glycosylation, are often required. However, a problem encountered in eukaryotic cell systems which have been transformed with heterologous genes suitable for the production of protein sequences destined for use, for example, as pharmaceuticals in humans, is that the glycosylation pattern on such proteins often acquires a native pattern, that is, of the eukaryotic cell system into which the protein has been introduced: glycosylated proteins are produced that comprise non-animal, that is to say, for example, non-mammalian glycosylation patterns and these in turn may be immunogenic and/or allergenic if applied in animals, such as mammals, e.g. humans.
Compared to mammalian-derived glycoproteins, plant-specific glycoproteins contain two additional residues. In the past, the use of recombinant glycoproteins produced by plants was limited by the plant-specific N-glycosylation that is acquired on such proteins. In the case of bryophytes Koprivova et al. ((2004), Plant Biotechnol J 2, 517-523) and in the case of seed plants Strasser et al. ((2004) FEBS Lett. 561, 132-136)) succeeded in overcoming this limitation using different approaches. The plants generated in the two studies showed complex N-glycosylation lacking the above mentioned two plant-specific sugar residues.
Moreover, in plants glycoprotein terminal beta 1,4-galactose residues are not found, indicating that a beta 1,4-galactosyltransferase is not present in plants. Stable integration and expression of this enzyme in tobacco plants (Bakker et al. (2001) Proc Natl Acad Sci USA 98, 2899-2904), in tobacco BY2 cells (Palacpac et al. (1999) Proc Natl Acad Sci USA 96, 4692-4697) as well as in gametophytic haploid bryophytes (Huether et al. (2005) Plant Biol 7, 292-299) has been described. The recombinant human beta 1,4-galactosyltransferase was functional and proteins isolated from transgenic material exhibited terminal beta 1,4-galactose residues.
The present invention is concerned with the further improvement of existing methods in order to ensure that polypeptides and proteins with still further improved functionality in animals, such as mammals, are produced.
The most complex N-glycan structures present on mammalian proteins, including human proteins, contain sialic acids as terminal sugar residues. Although the presence of sialylated glycoconjugates in non-transgenic suspension cultured cells of Arabidopsis thaliana was described recently by Shah et al. ((2003) Nat Biotechnol 21, 1470-1471), these results are under discussion (Seveno et al. (2004) Nat Biotechnol 11, 1351-1353).
However, the prior art does not provide any information on whether sialylation also takes place in bryophytes and may enable recombinant expression of heterologous glyco-proteins having the desired N-glycan characteristics. In addition, no data are available in the prior art as to the pure existence of sialic acid in any bryophyte.
A pre-requisite for sialylation on N-glycans is the presence of activated neuraminic acid (CMP NeuAc). In mammals different enzymes are involved in the synthesis of NeuAc (sialic acid)—the precursor of CMP NeuAc. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-6-kinase (genbank accession number: AF155663) is responsible for generating ManNAc-6P which is processed by N-acetylneuraminic acid phosphate synthase (genbank accession number: NM—018946) to NeuAc-9P. The enzyme responsible for processing NeuAc-9P into NeuAc is not described up to now. Activation of NeuAc takes place in the nucleus of mammalian cells. Responsible for generation of the activated sialic acid (CMP NeuAc) is the enzyme CMP-N-acetylneuraminic acid synthase (genbank accession number: NM—018686). The activated product has to be translocated from the nucleus into the Golgi apparatus—in this process the CMP-sialic acid transporter (genbank accession number: NM—006416) is involved. Finally, sialylation on N-glycans takes place by the transfer of CMP NeuAc on terminal sugar residues—e.g. 1,4 linked—galactose residues. For this purpose, expression of a sialyltransferase (e.g. alpha-2,6 sialyltransferase; accession number NM—003032, gene bank) has to be ensured. The bryophyte, Physcomitrella patens, a haploid non-vascular land plant, is able to be used for the production of recombinant proteins (WO 01/25456).
The life cycle of bryophytes is dominated by photoautotrophic gametophytic generation. The life cycle is completely different to that of higher plants wherein the sporophyte is the dominant generation and there are notably many differences to be observed between higher plants and bryophytes.
The gametophyte of bryophytes is characterised by two distinct developmental stages. The protonema which develops via apical growth, grows into a filamentous network of only two cell types (chloronemal and caulonemal cells). The second stage, called the gametophore, differentiates by caulinary growth from a simple apical system. Both stages are photoautotrophically active. Cultivation of protonema without differentiation into the more complex gametophore has been shown for suspension cultures in flasks as well as for bioreactor cultures (WO 01/25456). Cultivation of fully differentiated and photoautrophically active multicellular tissue containing only a few cell types is not described for higher plants. The genetic stability of the bryophyte cell system provides an important advantage over plant cell cultures. In cell cultures of higher plants the secondary metabolism is more differentiated and this results in differences in secondary metabolite profiles.
In addition, there are some important differences between bryophytes and higher plants on the biochemical level. Sulfate assimilation in Physcomitrella patens differs significantly from that in higher plants. The key enzyme of sulfate assimilation in higher plants is adenosine 5′-phosphosulfate reductase. In Physcomitrella patens an alternative pathway via phosphor-adenosine 5′-phosphosulfate reductase co-exists (Koprivova et al. (2002) J. Biol. Chem. 277, 32195-32201). This pathway has not been characterised in higher plants.
Further differences are reflected in the regeneration of the cell wall. Protoplasts derived from higher plants regenerate new cell walls in a rapid manner, independently of the culture medium. Direct transfer of DNA via polyethylene glycol (PEG) into protoplasts of higher plants requires pre-incubation at 4 to 10° C. to slow down the process of cell wall regeneration (U.S. Pat. No. 5,508,184). In contrast, cell wall regeneration of protoplasts derived from protonema of Physcomitrella is dependent on culture medium. Protoplasts can be cultivated without regeneration of the cell wall over long periods. Without the intention of being bound by theory, it appears that the secretion machinery of the protoplast, essential for cell wall regeneration and protein glycosylation, differs from that of higher plants. Moreover, Physcomitrella patens shows highly efficient homologous recombination in its nuclear DNA, a unique feature for plants, which enables directed gene disruption (Girke et al. (1998) Plant J 15, 39-48; Strepp et al. (1998) Proc Natl Acad Sci USA 95, 4368-4373; Koprivova (2002) J Biol Chem 277, 32195-32201; reviewed by Reski (1999) Planta 208, 301-309; Schaefer and Zryd (2001) Plant Phys 127, 1430-1438; Schaefer (2002) Annu. Rev. Plant Biol. 53, 477-501) further illustrating fundamental differences to higher plants.
It is an object of the present invention to provide a more efficient method of producing animal proteins comprising animal glycosylation patterns, and in particular, glycosylated human proteins comprising human glycosylation patterns thereon—containing sialic acid residues. It is a further object to provide an efficient process for the production of heterologous animal proteins comprising animal glycosylation patterns, particularly human proteins comprising human glycosylation patterns—containing sialic acid residues—in bryophytes, such as Physcomitrella patens. 
These and other objects will become apparent from the following description and examples provided herein.